Plants and methods for high density plant production

ABSTRACT

Provided are methods for growing a plant under certain stressed conditions that alter the morphology of the plant. In certain aspects, however, although the plant is altered and may be undesirable for commercial purposed, the plant still produces an adequate number of seeds for breeding purposes. Further, because plants may be smaller in size, they can be grown at higher densities, allowing the production of large populations of plants to be brought under controlled conditions which can exclude pollinating insects and thus increase the genetic purity achievable in a breeding program.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application62/435,209, filed on Dec. 16, 2016, which is incorporated by referenceherein in its entirety.

BACKGROUND

The goal of plant cultivation is generally to provide growingconditions, such as irrigation, light, spacing, temperature and accessto nutrients that promote healthy plant growth, including achieving acertain size and producing a number of seeds or fruit typical of ahealthy plant. For example for cotton, which is shrubby plant that growsto be about 3 to 5 feet tall, typical commercial plantings have a plantdensity of about 8 to 12 plants per square meter with rows spaced about1 meter apart. This spacing has been developed to optimize cropperformance by generally increasing yield per unit area and minimizingdisease susceptibility.

The large amount of space, however, needed to grow large populations ofplants such as cotton is an obstacle to growing plants in controlledenvironments, such as green houses or grow houses, where not only canconditions such as temperature, light, and water be controlled, but thepresence of pollinating insects minimized. This would be especiallyadvantageous in breeding programs where the genetic purity resultingfrom crosses can be increased due to the lack of interference frompollinating insects. Increases in genetic purity translate into higherefficiencies and/or lower costs in breeding programs. Further thesynchronization of flowering may also translate into higher efficienciesand/or lower costs in breeding programs by reducing overall cycle timeand resource waste.

Therefore, there remains a need for methods of growing plants for use inmore efficient breeding programs that are feasible in light of the spacelimitations.

SUMMARY

Provided herein are methods of producing a cotton seed by growing acotton plant from a cotton seed under a stressed condition. It isunderstood that reference to “an aspect” refers to any of the methodsdisclosed herein and should not be limited to any particular methodunless otherwise stated. In certain aspects, the stressed conditioncomprises growing the cotton plant in a volume of growth media of lessthan about 2,000 mL, less than about 1,500 mL, less than about 1,000 mL,less than about 500 mL, less than about 450 mL, less than about 350 mL,less than about 250 mL, or less than about 200 mL, for at least 40 days,for at least 50 days, for at least 60 days, for at least 70 days, for atleast 75 days, or for at least 80 days during the time from when thecotton seed germinates to when the cotton plant produces at least onecotton boll that contains a seed, wherein the seed comprises at leastone embryo with a cotyledon in the primordia stage and optionallywherein the seed comprises a black layer (restricted growth mediavolume). In certain aspects, the stressed condition comprises growingthe cotton plant at a temperature of at least about 37° C., 38° C., 39°C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., or48° C., for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hoursper day, for at least 40 days, for at least 50 days, for at least 60days, for at least 70 days, for at least 75 days, or for at least 80days during the time from when the cotton seed germinates to when thecotton plant produces at least one cotton boll that contains a seed,wherein the seed comprises at least one embryo with a cotyledon in theprimordia stage and optionally wherein the seed comprises a black layer(high temperature). In certain aspects, the stressed condition comprisesgrowing the cotton plant by providing less than about 500 mL, less thanabout 400 mL, less than about 300 mL, less than about 200 mL, less thanabout 100 mL, or less than about 50 mL of water per day on average, forat least 40 days, for at least 50 days, for at least 60 days, for atleast 70 days, for at least 75 days, or for at least 80 days during thetime from when the cotton seed germinates to when the cotton plantproduces at least one cotton boll that contains a seed, wherein the seedcomprises at least one embryo with a cotyledon in the primordia stageand optionally wherein the seed comprises a black layer (restrictedirrigation). In certain aspects, the stressed condition can be acombination of any of these.

In certain aspects, the method comprises growing a cotton plant from acotton seed under the stressed condition of: restricted growth mediavolume and/or restricted irrigation, wherein the cotton plant is alsogrown at a temperature of at least about 32° C., 33° C., 34° C., 35 °C., or 36° C. for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12hours per day, for at least 40 days, for at least 50 days, for at least60 days, for at least 70 days, for at least 75 days, or for at least 80days during the time from when the cotton seed germinates to when thecotton plant produces at least one cotton boll that contains a seed,wherein the seed comprises at least one embryo with a cotyledon in theprimordia stage and optionally wherein the seed comprises a black layer.

In certain aspects, the days during which the cotton plant is grownunder restricted growth media volume and/or restricted irrigationcorrespond to the days during which the plant is grown at a temperatureof at least about 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38°C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47°C., or 48° C., for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12hours a day. In certain aspects, the plant is grown at a nighttimetemperature of about 18° C. to 22° C. In certain aspects, on at leastabout 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% of the days on whichthe plant is grown at a temperature of at least about 37° C., 38° C.,39° C., 40° C., 41° C., 42° C., 43° C., 44° C., or 45° C., it is notgrown at this temperature for more than about 6, 7, 8, 9, 10, 11, 12,13, 14, 15, or 16, 18, or 20 hours per day. In certain aspects, on atleast about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% of the days onwhich the plant is grown at a temperature of at least about 37° C., 38°C., 39° C., 40° C., 41° C., 42° C., 43 ° C., 44° C., or 45° C., theplant is also grown at a temperature of about 18° C. to 22° C. for atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 hours per aday.

In certain aspects, the cotton plant is grown at least under the stresscondition of restricted growth media volume. In certain aspects, thecotton plant is grown at least under the stress condition of hightemperature. In certain aspects, the cotton plant is grown at leastunder the stress condition of restricted irrigation.

In certain aspects, the cotton plant grown under a stressed conditionand having produced at least one cotton boll that contains a seed,wherein the seed comprises at least one embryo with a cotyledon in theprimordia stage is of a diminished size, such as decreased in height,shortened internode length and/or having reduced biomass, compared to acontrol plant.

In certain aspects, the cotton plant is grown in a controlledenvironment.

In certain aspects, the cotton plant is grown in a volume of growthmedia of from any of about 200 mL, 250 mL, 300 mL, 350 mL, or 400 mL toany of about 300 mL, 350 mL, 400 mL, or 500 mL, for at least 40 days,for at least 50 days, for at least 60 days, for at least 70 days, for atleast 75 days, or for at least 80 days during the time from when thecotton seed germinates to when the cotton plant produces at least onecotton boll that contains a seed, wherein the seed comprises at leastone embryo with a cotyledon in the primordia stage and optionallywherein the seed comprises a black layer.

In certain aspects, the method further comprises applying a plant growthregulator (PGR) to the cotton plant before the first pinhead squarestage. In certain aspects, the method further comprises applying a PGRto the cotton plant at a day not past 20 days, 21 days, 22 days, 23days, or 25 days after germination. In certain aspects, the methodcomprises applying a PGR at 20 to 25 days after germination, 20 to 24days after germination, 21 to 23 days after germination, or 22 to 24days after germination. In certain aspects, the PGR is a compound knownto inhibit the synthesis, update, or persistence of gibberellic acid ina plant. Further, In certain aspects, the PGR is selected from the groupconsisting of chlormequat-CL, mepiquat-CL, AMO-1618, clorphonium-Cl,tetcylacis, ancymidol, flurprimidol, paclobutrazol, uniconazole-P,inabenfide, prohexadione-CA, trinexapac-ethyl, daminozide, exo-16,17-,dihydro-GA5-13-acetate, systemin, phytosulfokine, and rapidalkalinization factor. And, in certain aspects, the PGR is mepiquatchloride.

In certain aspects, the cotton plant having produced at least one cottonboll that contains a seed, wherein the seed comprises at least oneembryo with a cotyledon in the primordia stage, is less than about 30inches tall, less than about 24 inches tall, less than about 18 inchestall, less than about 15 inches tall, or less than about 12 inches tall.In certain aspects, the cotton plant having produced at least one cottonboll that contains a seed, wherein the seed comprises at least oneembryo with a cotyledon in the primordia stage, has at least 20 seeds,at least 30 seeds, at least 40 seeds, at least 50 seeds, or at least 60seeds.

In certain aspects, the seed of the cotton plant grown under a stressedcondition is used in a breeding program. Certain aspects comprisecollecting at least one seed from the plant grown under a stressedcondition. In certain aspects, the method further comprises testing atleast one collected seed or a plant grown from the collected seed. Thetesting can be, for example, genotyping and/or phenotyping for geneticand/or physical characteristics and traits. In certain aspects, themethod further comprises selecting a cotton plant to grow based on thetesting. In certain aspects, the method further comprises crossing acotton plant based on the testing.

In certain aspects, the cotton plant grown under a stressed condition isa member of a population of cotton plants grown at an average density ofat least 10, 30, 60, 75, 100, 125, or 150 plants per square meter. Incertain aspects, the population of plants comprises at least about 50cotton plants, at least about 100 cotton plants, at least about 500cotton plants, at least about 1,000 cotton plants, at least about 5,000cotton plants, at least about 10,000 cotton plants, at least about100,000 cotton plants, at least about 500,000 cotton plants, or at leastabout 1,000,000 cotton plants. Similarly, in certain aspects, thepopulation of plants comprises 50 or more cotton plants, 100 or morecotton plants, 500 or more cotton plants, 1,000 or more cotton plants,5,000 or more cotton plants, 10,000 or more cotton plants, 100,000 ormore cotton plants, 500,000 or more cotton plants, or 1,000,000 or morecotton plants. In certain aspects, the cotton plant grown under astressed condition is grown as a member of a population of cotton plantsat an average density of at least 10, 30, 60, 75, 100, 125, or 150plants per square meter, for at least 40 days, for at least 50 days, forat least 60 days, for at least 70 days, or for at least 80 days duringthe time from when the cotton seed germinates to when the cotton plantproduces at least one cotton boll that contains a seed, wherein the seedcomprises at least one embryo with a cotyledon in the primordia stage.In certain aspects, at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or99% of the cotton plants in the population have reaches a stage in theirlife cycle wherein pinhead squares have formed. In certain aspects, thepopulation of plants is grown in a controlled environment.

Provided herein are methods of producing a population of cotton plantsby: collecting at least one seed produced by a cotton plant grown undera stressed condition according to any of the aforementioned methods;testing (e.g., genotype and/or phenotype) at least one collected seed orplant grown from the collected seed; and growing and/or crossing acotton plant based on said testing to produce a population of cottonplants.

Provided herein are methods of breeding cotton plants by: collecting atleast one seed produced by a cotton plant grown under a stressedcondition according to any of the aforementioned methods. In certainaspects, the seed is tested (e.g., genotyped and/or phenotyped) todetermine the performance of, and/or a characteristic of, a parent plantand/or the offspring a plant produces.

Provided herein are cotton plants with an altered morphology. It isunderstood that reference to “an aspect” refers to any of the cottonplants with an altered morphology disclosed herein and should not belimited to any particular plant unless otherwise stated. In certainaspects, the cotton plant is less than about 30 inches, less than about24 inches, less than about 18 inches, less than about 15 inches, or lessthan about 12 inches, and wherein the plant has at least 20 seeds, atleast 30 seeds, at least 40 seeds, at least 50 seeds, or at least 60seeds. In certain aspects, the plant is grown according any of theaforementioned methods.

Provided herein are methods of growing a population of cotton plants,wherein the cotton plants have an altered morphology, by growing under astressed condition a population of cotton plants from cotton seeds. Asstated above, it is understood that reference to “an aspect” refers toany of the methods disclosed herein and should not be limited to anyparticular method unless otherwise stated. In certain aspects, thestressed condition comprises growing the cotton plants of the populationin an average volume per plant of growth media of less than about 2,000mL, less than about 1,500 mL, less than about 1,000 mL, less than about500 mL, less than about 450 mL, less than about 400 mL, less than about300 mL, less than about 250 mL, or less than about 200 mL, for at least40 days, for at least 50 days, for at least 60 days, for at least 70days, for at least 75 days, or for at least 80 days during the time fromthe average day of germination of the cotton seeds of the population tothe average day that the plants of the population produce at least onecotton boll that contains a seed, wherein the seed comprises at leastone embryo with a cotyledon in the primordia stage and optionallywherein the seed comprises a black layer (restricted growth mediavolume). In certain aspects, the stressed condition comprises growingthe population of cotton plant at a temperature of at least about 37°C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46°C., 47° C., or 48° C. for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,or 12 hours per day, for at least 40 days, for at least 50 days, for atleast 60 days, for at least 70 days, for at least 75 days, or for atleast 80 days during the time from the average day of germination of thecotton seeds of the population to the average day that the plants of thepopulation produce at least one cotton boll that contains a seed,wherein the seed comprises at least one embryo with a cotyledon in theprimordia stage and optionally wherein the seed comprises a black layer(high temperature). In certain aspects, the stressed condition comprisesgrowing the population of cotton plants by providing the cotton plantsof the population on average with less than about 500 mL, less thanabout 400 mL, less than about 300 mL, less than about 200 mL, less thanabout 100 mL, or less than about 50 mL of water per day on average, forat least 40 days, for at least 50 days, for at least 60 days, for atleast 70 days, for at least 75 days, or for at least 80 days during thetime from the average day of germination of the cotton seeds of thepopulation germinates to the average day that the plants of thepopulation produce at least one cotton boll that contains a seed,wherein the seed comprises at least one embryo with a cotyledon in theprimordia stage and optionally wherein the seed comprises a black layer(restricted irrigation). In certain aspects, the stressed condition canbe a combination of any of these.

In certain aspects, at least about 50%, 60%, 70%, 80%, 90%, or 95% ofthe plants in the population produce seed. In certain aspects, at leastone cotton plant of the population, having produced at least one cottonboll that contains a seed, wherein the seed comprises at least oneembryo with a cotyledon in the primordia stage and optionally whereinthe seed comprises a black layer, is of a diminished size, such asdecreased in height and/or having reduced biomass, compared to a controlplant. In certain aspects, at least about 50%, 60%, 70%, 80%, 90%, or95% of the plants in the population, having produced at least one cottonboll that contains a seed, wherein the seed comprises at least oneembryo with a cotyledon in the primordia stage and optionally whereinthe seed comprises a black layer, are of a diminished size, such asdecreased in height and/or having reduced biomass, compared to a controlplant.

In certain aspects, the population is grown in a controlled environment.

In certain aspects, at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or99% of the plants in the population have produced at least one cottonboll that contains a seed, wherein the seed comprises at least oneembryo with a cotyledon in the primordia stage and optionally whereinthe seed comprises a black layer, and are less than about 30 inchestall, less than about 24 inches tall, less than about 18 inches tall,less than about 15 inches tall, or less than about 12 inches tall. Incertain aspects, at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or99% of the plants in the population have at least 20 seeds, at least 30seeds, at least 40 seeds, at least 50 seeds, or at least 60 seeds.

In certain aspects, at least one plant from the population of plantsgrown under a stressed condition is used in a breeding program. Incertain aspects, the method further comprises collecting at least oneseed from at least one plant from the population of plants grown under astressed condition. In certain aspects, the method further comprisestesting (e.g., genotyping and/or phenotyping) at least one collectedseed or plant grown from the collected seed. In certain aspects, themethod further comprises growing and/or crossing a cotton plant based onthe testing.

In certain aspects, the population of cotton plants is grown at anaverage density of at least 10, 30, 60, 75, 100, 125, or 150 plants persquare meter. In certain aspects, the population of plants comprises atleast about 50 cotton plants, at least about 100 cotton plants, at leastabout 500 cotton plants, at least about 1,000 cotton plants, at leastabout 5,000 cotton plants, at least about 10,000 cotton plants, at leastabout 100,000 cotton plants, at least about 500,000 cotton plants, atleast about 1,000,000 cotton plants. Similarly, in certain aspects, thepopulation of plants comprises 50 or more cotton plants, 100 or morecotton plants, 500 or more cotton plants, 1,000 or more cotton plants,5,000 or more cotton plants, 10,000 or more cotton plants, 100,000 ormore cotton plants, 500,000 or more cotton plants, or 1,000,000 or morecotton plants. In certain aspects, the population of cotton plants isgrown under a stressed condition at an average density of at least 10,30, 60, 75, 100, 125, or 150 plants per square meter, for at least 40days, for at least 50 days, for at least 60 days, for at least 70 days,or for at least 80 days during the time from the average day ofgermination of the cotton seeds of the population germinates to theaverage day that the plants of the population produce at least onecotton boll that contains a seed, wherein the seed comprises at leastone embryo with a cotyledon in the primordia stage and optionallywherein the seed comprises a black layer.

Provided herein are populations of cotton plants with an alteredmorphology. It is understood that reference to “an aspect” refers to anyof the populations of plants disclosed herein and should not be limitedto any particular population unless otherwise stated. In certainaspects, the population of plants is grown at a density of at least 30,60, 65, 75, 100, 125, or 150 plants per square meter. In certainaspects, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or99% of the plants in the population have reached a stage in their lifecycle when pinhead squares have formed. In certain aspects, at leastabout 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the plants in thepopulation produce at least one cotton boll that contains a seed,wherein the seed comprises at least one embryo with a cotyledon in theprimordia stage and optionally wherein the seed comprises a black layer.In certain aspects, at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99%of the plants in the population produce at least 20 seeds, at least 30seeds, at least 40 seeds, at least 50 seeds, or at least 60 seeds. Incertain aspects, at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% ofthe plants in the population are less than about 30 inches tall, lessthan about 24 inches tall, less than about 18 inches tall, less thanabout 15 inches tall, or less than about 12 inches tall. In certainaspects, the average height of the population is less than about 30inches tall, less than about 24 inches tall, less than about 18 inchestall, less than about 15 inches tall, or less than about 12 inches tall.In certain aspects, the population of plants comprises at least about 50cotton plants, at least about 100 cotton plants, at least about 500cotton plants, at least about 1,000 cotton plants, at least about 5,000cotton plants, at least about 10,000 cotton plants, at least about100,000 cotton plants, at least about 500,000 cotton plants, at leastabout 1,000,000 cotton plants. Similarly, in certain aspects, thepopulation of plants comprises 50 or more cotton plants, 100 or morecotton plants, 500 or more cotton plants, 1,000 or more cotton plants,5,000 or more cotton plants, 10,000 or more cotton plants, 100,000 ormore cotton plants, 500,000 or more cotton plants, or 1,000,000 or morecotton plants.

In certain aspects, the population of plants is grown according to anyof the aforementioned methods. In certain aspects, the population ofplants is grown in a controlled environment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows two different cotton plants with cotton bolls (seeds) andan altered morphology. The cotton plants were grown in approximately 380mL of soil and have a reduced stature, as evident in relationship to thehand holding it (left) and measuring tape showing 12 inches (right).

FIG. 2 shows cotton plants with reduced stature in comparison to plantsof normal height.

FIG. 3 shows cotton plants of reduced stature with bolls, grown at highdensity in a tray (top), in comparison to large, field grown plants(bottom).

FIG. 4 illustrates examples of a time period, e.g. “at least 40 days,”“at least 60 days,” and “at least 80 days,” during the time from whenthe cotton seed germinates to when the cotton plant produces at leastone cotton boll that contains a seed as described in detail herein.Consecutive as well as non-consecutive days are exemplified.

FIG. 5 shows distribution of flowering time as days after planting (DAP)in a population of cotton plants for several cotton lines.

FIG. 6 shows the results disclosed in FIG. 5 as the cumulative % ofplants that have flowered at DAP.

DETAILED DESCRIPTION

Provided herein are methods of cultivating plants, such as crop plants,to produce reproductively-viable (e.g., seed-producing) plants yet withaltered morphologies amendable for growth at high density and/or use inbreeding programs, and in particular for growth under controlledconditions, e.g., in a controlled environment. In certain aspects, thealtered morphology is a miniaturized plant such as a plant having areduced stature, decrease in height, shortened internode length, reducedbiomass, and the like. Such plants can be grown at higher densities andthus require less space for a given number of plants. This in turn makesit more feasible to grow a population of plants under cover (i.e.,generally protected from pollinating insects), which in turn canincrease the genetic purity of crosses achieved in a breeding program,making the breeding program more efficient and/or cost effective. Thus,in certain aspects, the methods are performed for the purpose ofimproving the efficiency breeding programs. Further, it has beendiscovered populations of plants grown under stressed conditionsdisclosed herein can display tighter synchronization in theirdevelopment time, such as flowering and/or seed production, which alsocan improve the efficiency breeding programs.

Methods include subjecting plants to one or more stressed conditionsand/or application of certain plant growth regulators (PRGs). Furtherprovided are seed producing plants with altered morphologies compared tocorresponding control plants.

While in certain aspects, a plant grown under the conditions of stressand/or application of a PGR disclosed herein produces seed, the numberof seed need not be as many as a plant grown under normal conditions orwanted for commercial sale. For example, in a plant breeding program,only a certain number of seeds may be needed for screening andreplanting, and thus any seeds in excess of that amount are unnecessary.The purpose of breeding programs is to cross plants and recover seedsfrom those crosses in order to capture and analyze the genetics of theseeds and/or to recover the seed so that that resulting plant and itsgenetics can be used in future crosses. The more efficiently that thisprocess can be done, the more quickly new lines of plants can bedeveloped and/or the lower the costs of development. It is currentlythought that the most efficient way to grow plants in a breeding programis to grow plants in a manner that maximizes health and growth, e.g.,that mimic commercial planting conditions. However, the number of seedsrequired for use in a breeding program may be less than the amount ofseed typically produced by commercial growers attempting to maximizecotton fiber yield. In fact, producing extra seeds at certain steps of abreeding program could be considered waste. Thus, it may be acceptableat certain steps of a breeding program to grow plants that produce afewer number of seeds than their genetic potential when grown underoptimal or commercial planting conditions.

Definitions

To the extent necessary to provide descriptive support, the subjectmatter and/or text of the appended claims is incorporated herein byreference in their entirety.

It will be understood by all readers of this written description thatthe exemplary embodiments described and claimed herein may be suitablypracticed in the absence of any recited feature, element or step thatis, or is not, specifically disclosed herein.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “a plant,” is understood to represent oneor more plants. As such, the terms “a” (or “an”), “one or more,” and “atleast one” can be used interchangeably herein. The phrase “one or more”as used herein is interchangeable with “at least one.”

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term and/or” as used in a phrase such as “Aand/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that wherever aspects are described herein with thelanguage “comprising,” otherwise analogous aspects described in terms of“consisting of” and/or “consisting essentially of” are also provided.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. Numeric ranges areinclusive of the numbers defining the range. Even when not explicitlyidentified by “and any range in between,” or the like, where a list ofvalues is recited, e.g., 1, 2, 3, or 4, the disclosure specificallyincludes any range in between the values, e.g., 1 to 3, 1 to 4, 2 to 4,etc.

The headings provided herein are solely for ease of reference and arenot limitations of the various aspects or aspects of the disclosure,which can be had by reference to the specification as a whole.Accordingly, the terms defined immediately below are more fully definedby reference to the specification in its entirety.

A used herein, a “plant” refers to a whole plant or any part thereof.For example, for purposed of this disclosure, applying a plant growthregulator to a “plant” includes applying the plant growth regulator to awhole plant or any part thereof or plant part (e.g., to the leaves,stems, roots, seeds, etc.).

As used herein, a “population of plants,” “plant population,” or thelike refers to at least two or more plants. Most commonly, the termsrelate to a breeding population of plants from which members areselected and crossed to produce progeny in a breeding program. Apopulation of plants can include the progeny of a single breeding crossor a plurality of breeding crosses, and can be either actual plants orplant derived material, or in silico representations of the plants. Thepopulation members need not be identical to the population membersselected for use in subsequent cycles of analyses or those ultimatelyselected to obtain final progeny plants. Often, a plant population isderived from a single biparental cross, but may also derive from two ormore crosses between the same or different parents. Although apopulation of plants may comprise a small plurality of individuals,those of skill in the art will recognize that plant breeders commonlyuse population sizes ranging from one or two hundred individuals toseveral thousand, and that the highest performing 5-20% of a populationis what is commonly selected to be used in subsequent crosses in orderto improve the performance of subsequent generations of the population.

As used herein, the term “non-naturally occurring” condition, substance,composition, entity, plant, organism, individual, and/or any combinationthereof, or any grammatical variants thereof, is a conditional term thatexplicitly excludes, but only excludes, those forms that arewell-understood by persons of ordinary skill in the art as being“naturally-occurring,” or that are, or might be at any time, determinedor interpreted by a judge or an administrative or judicial body to be,“naturally-occurring.”

As used herein, the terms “flower” and “inflorescence” are usedinterchangeably.

The methods described herein are not limited to the process by which apopulation of plants is brought together to be cultivated. For example,it is commonly understood that seeds form after fertilization andcomprise a zygote or embryo surrounded by tissues that were once part ofthe ovule. However, the term “seed” as used herein can also have a moregeneral meaning to include any viable plant cell capable of undergoingcell division (e.g. mitosis) to grow, and/or develop, and, in certainembodiments, form reproductive structures. A seed can also be anythingthat can be sown, or planted, directly into growth media includingvegetative structures, like potato tubers (“seed” potatoes), and/orsugarcane stalks, and/or cells of a callus. A seed may also comprise azygote or embryo encased in some form of protective layer. In certainembodiments the protective layer comprises tissues derived from plantcells (e.g. cells of the maternal parent ovule), and a seed may or maynot include other tissues, for example, when the layers of the seedprotecting or enclosing the embryo are technically considered to be partof a dried fruit or kernel, as they are in corn and sunflower. Evenartificial methods of coating or protecting an embryo with a wide rangeof chemicals that serve at least the purpose of protecting and/orsustaining, and or otherwise supporting the embryo are envisioned. Aseed could even be a naked embryo, provided one was able to culture itinto a vegetative or flowing plant using one or more of the methodsdescribed herein. One of skill will immediately appreciate that themethods disclosed herein could be used in conjunction with any otherprocess that brings about a population of plants growing in a controlledenvironment in the high densities and/or in at least one of the stressedconditions described herein.

As used herein, the term “elite,” “elite plant,” and the like describesa group, germplasm, or population of at least one crop plant that hasresulted from human-directed breeding and selection for superioragronomic performance. An “elite population” is an assortment of eliteindividuals or lines that can be used to represent the state of the artin terms of agronomically superior genotypes of a given crop species,such as cotton. Similarly, an “elite germplasm” or “elite strain ofgermplasm” is an agronomically superior germplasm, typically derivedfrom and/or capable of giving rise to a plant with superior agronomicperformance, such as an existing or newly developed elite line ofcotton. In contrast, an “exotic plant,” “exotic line,” or “exoticgermplasm” is a plant, line, or germplasm derived from a plant notbelonging to an available elite line or strain of germplasm. In thecontext of a cross between two plants or lines of germplasm, an exoticgermplasm is not closely related by descent to the elite germplasm withwhich it is crossed. Most commonly, the exotic germplasm is not derivedfrom any known elite line of a crop, but rather is selected to introducegenetic elements (typically desired alleles) into a breeding program.

As used herein, a “control” plant is a plant (or a member of apopulation of plants) recognized as having a representative phenotype(e.g., number of inflorescences, number of seeds, height, internodelength, biomass, and the like), of a plant grown under normallyrecommended growing conditions, but that is in other respects, such asgenetic makeup, comparable to a plant grown under a stressed condition.For example, one of ordinary skill in the art would understand a controlplant to have one or more of the following attributes: results from aseed derived from the same haploid induction cross; has at least oneparent in common with the treated plant; shares a common ancestor withthe treated plant within twelve generations; shares sufficient commongenetic heritage with the treated plant that one of ordinary skill inthe art of plant breeding would recognize the control plant as a validcomparison for establishing a correlation between the stressed growthcondition and the resulting phenotype; and/or achieves a morphologyconsidered typical of the mature plant. One of ordinary skill in the artwill recognize that a control plant that by chance (e.g., a statisticaloutlier), by some other type of manipulation, or other reason comprisesa phenotype that varies from a representative phenotype of controlplants would not be an appropriate control plant for comparison.

As used herein, the term “grown in a controlled environment,” means thata plant or population of plants is grown in an enclosure capable ofexcluding pollinating insects. Such enclosures include screened inenclosures such as cages, green houses, hothouses, grow houses, hoophouses, warehouses, and growth chambers. It is understood that “capableof excluding pollinating insects,” does not mean that all pollinatinginsects are absolutely excluded, but that fewer pollinating insects haveaccess to the plant or population of plants than if the plant orpopulation of plants was not grown in a controlled environment. Growthin a controlled environment may also include control over one or moreadditional factors such as lighting, temperature, humidity, irrigation,growth media volume, protection from the elements such as wind and hail,exclusion of windborne pollen, and protection from windblown pesticidecontamination.

A plant “growth media” as referred to herein can be any type ofsubstance suitable for growing plants. Commonly, soil is used as agrowth media for growing plants.

A flower bud of a cotton plant can be referred to as a “square”. Itgenerally comprises a central corolla containing re-productivestructures (e.g. anthers) and sepals and surrounded by three (orsometimes four) bracts. Several morphological stages of squaredevelopment (i.e. “squaring”) are recognized by cotton growers. The“pinhead” square stage begins when the square first becomes identifiableto the naked eye and it is followed by the “match-head” or “one-thirdgrown” square. Later, the bud takes on a candle shape, typically justbefore blooming.

Stressed Conditions

Disclosed herein are methods including growing plants, producing seeds,and conducting plant breeding, and the like, in which certain stressesare applied to the plants. These stressed conditions deviate from theestablished conditions recommended for commercial production orconditions that would likely be tested in research programs aimed atoptimizing growth conditions for maximum plant health and yield. Incertain aspects, one or more of these stressed conditions is the resultof human control and/or manipulation of the growing conditions(“controlled conditions,” e.g., in a “controlled environment”) and varyfrom conditions that may naturally be experienced by plants grown in thefield. That is, even though plants in the field may experienceconditions of e.g., drought and/or high temperatures due to naturalweather conditions, certain aspects of the methods disclosed herein aredistinguishable from those conditions because the conditions are beingcontrolled and/or manipulated by humans, such as within an enclosure orby planting in a restricted amount of growth medium and/or growingplants to maturity at high density.

In certain aspects, a method involves growing a plant from a seed. Incertain aspects, application of one or more stressed conditions resultsin an altered plant morphology compared to a control plant. In certainaspects, one or more of these stressed conditions can be applied fromwhen the seed is sown and/or germinated and maintained throughout all ora portion of the period during which a plant is grown. Further incertain aspects, a plant with an altered morphology and despite thestressed conditions, still produces at least some seed that can be used,for example, in a breeding program.

While the details of different stressed conditions are describedseparately below, it is understood that methods herein can comprise anyone, any combination of, or all of the stressed conditions. Further,while the stressed conditions may be described as applied to a plant, itis understood that any of such stressed condition or combination ofstressed conditions can be applied to a population of plants.

Restricted Growth Media Volume

In certain aspects, the stressed condition applied to a plant is growingthe plant in a restricted volume of growth media that is reduced, insome instances significantly from the generally recommended amount, toachieve altered plant morphologies. In certain aspects, plants grownunder restricted soil volume stress (or a combination of soil volumestress with other types of stressed conditions and/or the application ofplant growth regulators) resulting in altered morphologies still produceat least some seed.

Cotton plants, for example, are commonly grown spaced apart in openfields or if in containers, in containers with at least about 3 L ofsoil volume, e.g., 3 L buckets or bags, to allow them what is generallyconsidered sufficient room for healthy growth. In contrast, in certainaspects of the methods herein, a restricted soil volume is less thanabout 2,000 mL, less than about 1,500 mL, less than about 1,000 mL, lessthan about 900 mL, less than about 800 mL, less than about 700 mL, lessthan about 600 mL, less than about 500 ml, less than about 450 mL, lessthan about 400 mL, less than about 350 mL, less than about 300 mL, lessthan about 250 mL, or less than about 200 mL of soil volume. In certainaspects, a restricted soil volume is from any of about 100 mL, 200 mL,300 mL, 400 mL, 450 mL, 500 mL, or 1,000 mL of soil volume to any ofabout 300 mL, 400 mL, 450 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL,1,000 mL, 1,500 mL, or 2,000 mL of soil volume. For example, from about200 mL to about 500 mL, from about 250 mL to about 450 mL, from about300 mL to about 500 mL, or from about 350 mL to about 450 mL of soilvolume.

It is understood that the amount of soil that a plant is grown in can berestricted by growing the plant in a container. In certain aspects, oneplant is grown per container. More than one plant, however, can be grownin a shared container. In such cases, the amount of soil volume perplant can be considered the amount of soil volume in the containerdivided by the number of plants in the container.

It may be common practice to sow a seed in a small volume of soil andthen replant the plant into a larger container containing more soil, oreven a series of successively larger containers, as the plant grows andbecomes larger. If the plant is replanted as it grows into an amount ofsoil that does not create stress, it will not achieve the morphologiesdescribed herein. In contrast, in the methods disclosed herein, a plantis grown in a restricted soil volume for a defined period of time sothat the plant is stressed as it grows. In certain aspects, a cottonplant is grown in a restricted soil volume for at least 40 days, atleast 50 days, at least 60 days, at least 70 days, at least 75 days, atleast 80 days, or at least 85 days during the time from when the seedgerminates to when the plant produces at least one cotton boll thatcontains a seed, wherein the cotton seed comprises at least one embryowith a cotyledon in the primordia stage. In certain aspects, the cottonseed comprises a black layer. Under permissive conditions forgermination, a cotton seed, for example, will germinate generally around12 hours to 48 hours after being sown. Similarly, in certain aspects, anon-cotton plant is grown in a restricted soil volume for acorresponding percentage of days during the time from when the seedgerminates until when the plant reaches its reproductive stage. Thenumber of days during which the plant is grown under the stressedcondition need not be consecutive, i.e., the number of days in therestricted soil volume is the cumulative number of days from when theseed is sown or germinates to when the plant reaches reproductive stage.In certain aspects, the period of time is in consecutive days.

Cultivation at High Temperatures

In certain aspects, plants are stressed by cultivation at hightemperatures. Without being bound by theory, it is suggested in the artthat temperatures above about 35° C. can hamper photosynthesis andadditionally, plant development and survival. In certain aspects,cultivation at high temperatures is combined with the aforementionedmethods of growing plants in restricted soil volumes and/or with thebelow mentioned methods of growing plants with restricted irrigation. Incertain aspects, the temperatures are at least about 5° C. higher thanwhat would normally be recommended as the highest temperature at whichto grow the plant.

It has been discovered that growing a plant at daily high temperaturesin the range of about 37° C. to about 48° C., even for just a few hoursa day, can result in altered plant morphologies. In certain aspects,plants that are grown under high temperature stress (or a combination ofhigh water stress with other types of stressed conditions and/or theapplication of plant growth regulators) resulting in alteredmorphologies still produce at least some seed.

In certain aspects, a plant is grown at a temperature of at least about37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C.,46° C., 47° C., or 48° C. In certain aspects, the duration of time perday during which a plant is grown at the temperature can be as little as2 hours, or at least about 2 hours, 3 hours, 4 hours, 5 hours, 6 hours,7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours per day. Theduration of time per day during which a plant is exposed to the abovetemperatures can be consecutive and/or cumulative over the course of aday, including non-daylight hours. Generally, however, the temperaturerises during the day and then decreases overnight. In certain aspects,plants are grown at cooler temperatures overnight, such as at least 5°C., 10° C., 15° C., 20° C., or 25° C. cooler than the maximum daytimetemperature. In certain aspects, the average nighttime temperature isfrom about 18° C. to about 22° C. In certain aspects, a cotton plant isgrown at these high temperatures and times for at least 40 days, atleast 50 days, at least 60 days, at least 70 days, at least 75 days, atleast 80 days, or at least 85 days during the time from when the seedgerminates to when the plant produces at least one cotton boll thatcontains a seed, wherein the cotton seed comprises at least one embryowith a cotyledon in the primordia stage. In certain aspects, the cottonseed comprises a black layer. Similarly, in certain aspects, anon-cotton plant is grown at these high temperatures and times for acorresponding percentage of days during the time from when the seedgerminates until when the plant reaches its reproductive stage. Thenumber of days during which the plant is grown under the stressedcondition need not be consecutive, i.e., the number of days the plant isgrown at a high temperature is the cumulative number of days from whenthe seed is sown or germinates to when the plant reaches reproductivestage. For example, a plant can be grown at a high temperature for a fewdays, then at a lower temperature for a day or more, and then back tobeing grown at a high temperature, and so forth. In certain aspects, theperiod of time is in consecutive days.

In certain aspects, the duration of time that a plant is subjected tohigh heat is limited to prevent overstressing the plant to a point thatit will not produce seeds. Thus, in certain aspects, on at least about50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% of the days on which theplant is grown at a temperature of at least about 37° C., 38° C., 39°C., 40° C., 41° C., 42° C., 43° C., 44° C., or 45° C., it is not grownat this temperature for more than about 6, 7, 8, 9, 10, 11, 12, 13, 14,15, or 16, 18, or 20 hours per day, i.e., grown below this temperaturefor at least 4, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 hours perday. In certain aspects, on at least about 50%, 60%, 70%, 80%, 90%, 95%,98%, or 100% of the days on which the plant is grown at a temperature ofat least about 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C.,44° C., or 45° C., the plant is also grown at a temperature of about 18°C. to 22° C. for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, or 16 hours per a day.

A combination of stressed conditions may produce an additive effect inwhich the severity of an individual stressed condition may be reducedbut still achieve a plant with a desired altered morphology. In certainaspects, when a plant is grown under restricted growth media volumeand/or restricted irrigation stressed conditions as described elsewhereherein, the high temperature stressed condition can be reduced to atleast about 32° C., 33° C., 34° C., 35° C., or 36° C., under theconditions of hours per day, days between germination and seedformation, etc., otherwise described herein.

As described anywhere herein unless otherwise stated, while the daysbetween germination and seed formation, reaching reproductive stage, andthe like during which various stressed conditions are applied willgenerally overlap, it is not necessary that they correspond or evenoverlap. For example, a plant could be subjected to limited irrigationon one day at a normal temperature and then given a normal amount ofwater the next day but subjected to high temperatures. For example, aplant may be grown in a restricted soil volume for a period of timeunder normal water and temperature conditions, and then replanted in anormal soil volume but subjected to temperature and/or irrigationstress.

Restricted Irrigation

In certain aspects, plants are stressed by restricting the amount ofwater that they receive. In certain aspects, water restriction iscombined with the aforementioned methods of growing plants in restrictedsoil volumes and/or high temperatures. For example, whereas generallyrecommended irrigation of a plant may be about 500 mL to about 1,000 mLof water per 24 hour period for most growing days, in certain aspects, aplant is provided with about 1/10^(th) or less water, such as less thanabout 100 mL of water, during the same period of time. In certainaspects, plants are grown under water restriction (or a combination ofrestricted irrigation stress with other types of stressed conditionsand/or the application of plant growth regulators) resulting in alteredmorphologies still produce at least some seed.

In certain aspects, the amount of restricted water is not determined asa daily basis, but rather the average amount of water per day over agiven number of days. For example, a plant that is watered with 600 mLon a first day and then not watered the next two consecutive days wouldon average have been water with 200 mL of water per day during the threeday period.

In certain aspects, a plant is provided with less than about 500 mL,less than about 400 mL, less than about 300 mL, less than about 200 mL,less than about 100 mL, or less than about 50 mL of water per day onaverage. In certain aspects, a plant is provide with any of about 1 mL,10 mL, 25 mL, 50 mL, 100 mL, 200 mL, 250 mL, or 300 mL of water per dayon average, to any of about 50 mL, 100 mL, 200 mL, 250 mL, 300 mL, 400mL, or 500 mL of water per day on average. In certain aspects, a cottonplant is grown under any of these restricted irrigation conditions forat least 40 days, at least 50 days, at least 60 days, at least 70 days,at least 75 days, at least 80 days, or at least 85 days during the timefrom when the seed germinates to when the plant produces at least onecotton boll that contains a seed, wherein the cotton seed comprises atleast one embryo with a cotyledon in the primordia stage. In certainaspects, the cotton seed comprises a black layer. Similarly, in certainaspects, a non-cotton plant is grown under restricted irrigationconditions for a corresponding percentage of days during the time fromwhen the seed germinates until when the plant reaches its reproductivestage.

Plant Growth Regulators (PGRs)

Certain aspects provide for the use of plant growth regulators (PGRs) toinduce altered plant morphologies, either by the use of PGRs alone or incombination with one or more of the aforementioned stressed conditionsto achieve an altered morphology. In certain aspects, plants treatedwith a PGR (or a combination of PGR with one or more aforementionedstresses) resulting in altered morphologies still produce at least someseed.

In certain aspects provided herein, a plant can be contacted with a widevariety of PGRs. PGRs are a class of compounds that affect the cellularprocesses, growth, development or behavior of a plant. PGR can beresponsible for accelerating or retarding the rate of growth ormaturation or otherwise altering the behavior of a plant. In someaspects, a PGR is a naturally-occurring plant hormone capable ofaltering flower development, internode length, apical dominance,ripening, root architecture, fruiting and/or other characteristics of aplant. In some aspects, a PGR is a chemical capable of altering flowerdevelopment, internode length, apical dominance, ripening, rootarchitecture, fruiting and/or other characteristics of a plant. Plantgrowth regulators include auxins (e.g. IAA) and auxin inhibitors,cytokinins (e.g. BAP) and cytokinin inhibitors, compounds that canstimulate ethylene production (i.e. ACC and the like) and compounds thatcan inhibit ethylene production (AVG and the like), and compounds thatinhibit ethylene perception (silver and the like). Plant growthregulators also comprise compounds that modulate plant perception,signaling, and/or behavior, such as giberrellins and their inhibitors(e.g. Paclobutrazol (PBZ) or uniconazole), abscisic acid and itsinhibitors, and jasmonic acid and its inhibitors. In certain aspects,the plant growth regulator is a plant hormone, gibberellic acidinhibitor, cytokinin, or any combination thereof.

In certain aspects, a PGR is an exogenously-provided compound that canbe introduced to the surface of a plant and migrate into a plant tissue.In some aspects, the PGR acts extracellularly within the plant tissue,such as interacting with receptors on the outer cell surface. In someaspects, the PGR enters into cells within the tissue. In some aspects,the PGR is contained within a liquid. Such liquids include, but are notlimited to aqueous and non-aqueous solutions, suspensions, emulsions,and colloidal dispersions. A “plant treatment solution” or “treatmentsolution” can refer to any solution of liquid that comprises a PGR.

In certain aspects, contact between the plant and the treatment agent isachieved by dipping, submerging, or otherwise inserting the plant into areservoir of liquid comprising the plant treatment agent. Other methodsof contacting a plant with a treatment agent include spraying or mistingthe plant with a solution comprising a plant treatment agent oragitating or tumbling a plant in a solution comprising a plant treatmentagent. In certain aspects, contact between the plant and the treatmentagent is achieved by a soil drench, which comprises adding a liquidtreatment agent to the soil or growth medium near the plant roots.

In certain aspects, liquids are of an aqueous nature. In certainaspects, aqueous liquids can comprise water soluble components. Incertain aspects, aqueous liquids can comprise water insolublecomponents, can comprise an insoluble component that is made soluble inwater by addition of a surfactant, or can comprise any combination ofsoluble components, insoluble components, and surfactants.

In certain aspects, the PGR can be any compound known to inhibit thesynthesis, uptake, or persistence of gibberellic acid in a plant. Forexample, one non-limiting example of a PGR that can be used with thisinvention is selected from the group comprising chlormequat-CL,mepiquat-CL, AMO-1618, clorphonium-Cl, tetcylacis, ancymidol,flurprimidol, paclobutrazol, uniconazole-P, inabenfide, prohexadione-CA,trinexapac-ethyl, daminozide, exo-16,17-, and dihydro-GA5-13-acetate.Other examples include peptide hormones, for example, systemin,phytosulfokine, rapid alkalinization factor. In certain aspects, the PGRis mepiquat chloride.

IAA is indole-3-acetic acid, and IBA is inodole-3-butyric acid. Both arenaturally-occurring forms of a class of plant hormones called auxins.Other variations of auxin can be used, including synthetic auxins, suchas 2,4-D (2,4-Dichlorophenoxyactic acid and □-NAA (□-Naphthalene aceticacid).

As used herein, PBZ is paclobutrazol,(2S,3S)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol, also written as C15H1OCIN30, a plant growth regulator andtriazole fungicide. It is a known antagonist of the plant hormonegibberellins that inhibits giberellin biosynthesis, reducing internodalgrowth and increasing stem girth. BAP is 6-Benzylaminopurine,N-(Phenylmethyl)-7H-pruin-6-amine, also written as C12H11N5. IAA isindole-3-acetic acid, and IBA is inodole-3-butyric acid. Both arenaturally-occurring forms of a class of plant hormones called auxins.Other variations of auxin can be used with this invention, includingsynthetic auxins, such as 2,4-D (2,4-Dichlorophenoxyactic acid) and1-NAA (1-Naphthalene acetic acid).

As used herein, uniconazole is(e)-(+/−)-beta-((4-chlorophenyl)methylene)-alpha-(1,1-dimethylethyl)-1h-1,2,4-triazole-1-ethanol,also written as C15H18CIN3O, also known as uniconazole-P. It is atriazole-type plant growth retardant and known antagonist of the planthormone giberellin that reduces internodal growth and increases stemgirth.

As used herein, mepiquat chloride is also known asN,N-dimethylpiperidinium chloride, C7H16CIN, PIX®, mepiquat-CL.

In general, PGRs used herein will be water soluble agents. However, theuse of PGRs with high, intermediate, low or negligible water solubilitycan, in certain aspects, be facilitated by the use of liquidcompositions that also comprise various transfer or conditioning agents.Transfer or conditioning agents can comprise any agent that facilitatesmigration of plant treatment agents to the plant (e.g., plant cells)and/or that facilitate uptake of plant treatment agents by the plant.Transfer or conditioning agents include, but are not limited to, (a)surfactants, (b) an organic solvents or an aqueous solutions or aqueousmixtures of organic solvents, (c) oxidizing agents, (d) acids, (e)bases, (f) oils, (g) enzymes, or combinations thereof. In certainaspects, methods can optionally include an incubation step, aneutralization step (e. g., to neutralize an acid, base, or oxidizingagent, or to inactivate an enzyme), a rinsing step, or combinationsthereof whereby the liquid and plant treatment agent contained thereinis treated either before or after delivery to the plant. Transfer orconditioning agents thus include, but are not limited to, emulsions,reverse emulsions, liposomes, and other micellar-like compositions.Examples of useful adjuvants include surfactants and effective moleculescontained therein, which include sodium or lithium salts of fatty acids(such as tallow or tallowamines or phospholipids). Transfer orconditioning agents can comprise salts including, but not limited to,sodium, ammonium, calcium, lithium, magnesium, chloride, sulfide, andsulfate salts. Certain aspects of the methods provided herein usecounter-ions or other molecules that are known to associate with planttreatment agents. For certain negatively charged plant treatment agentssuch as polynucleotides, cations such as inorganic ammonium ions, alkylammonium ions, lithium ions, polyamines such as spermine, spermidine, orputrescine, and the like can be used. Organic solvents useful inconditioning a plant cell to permeation with certain plant treatmentagents including, but not limited to polynucleotides, are solvents suchas DMSO, DMF, pyridine, N-pyrrolidine, hexamethyiphosphoramide,acetonitrile, dioxane, polypropylene glycol, or other solvents that aremiscible with water. Naturally derived or synthetic oils with or withoutsurfactants or emulsifiers can be used, e. g., plant-sourced oils, cropoils (such as those listed in the 9th Compendium of Herbicide Adjuvants,publicly available on the world wide web (internet) at“herbicide.adjuvants.com”) can be used. Oils useful in certain liquidcompositions used in the methods provided herein include, but are notlimited to, paraffinic oils, polyol fatty acid esters, or oils withshort-chain molecules modified with amides or polyamines such aspolyethyleneimine or N-pyrrolidine.

In certain aspects of any of the methods herein, a plant is contactedwith the PGR by drenching, gassing, injecting, or spraying.

In certain aspects, a broad range of chemical concentrations can be usedin conjunction with these methods and one of ordinary skill in the artcan optimize the dose administered to a given plant subjected to any ofthe stressed conditions disclosed herein in order to maximize a desiredmorphology.

In certain aspects, the use of the PGR is inconsistent with priormethods of applying the PGR. For example, it is expressly recommended bythe manufacturer that a user should not apply mepiquat chloride untilafter pinhead squares have formed on the plant (typically 25-30 daysafter planting (DAP)). In certain aspects, however, the method comprisesapplying the PGR before the first pinhead square stage. In certainaspects, the method comprises applying the PGR at a day not past 20days, 21 days, 22 days, 23 days, or 25 days after germination. Incertain aspects, the method comprises applying a PGR at 20 to 25 daysafter germination, 20 to 24 days after germination, 21 to 23 days aftergermination, or 22 to 24 days after germination.

Altered Plant Morphologies

Provided herein are plants with altered morphologies due to theapplication of one or more stressed conditions and/or application of aPGR while the plant is growing. That is, a plant with an alteredmorphology disclosed herein grown according to any of the aforementionedmethods. In particular, the aforementioned growing conditions result inplants that typically have a reduced height, shorter internode lengths,and/or decreased biomass than plants grown under normal or recommendedgrowing conditions. While the growth of these plants may appear sicklyor stunted to one of ordinary skill in the art, it has been discoveredthat under the right conditions, such plants can produce at least someseeds and thus can be useful, for example, in plant breeding programs.In certain aspects, a plant is grown in a controlled environment, forexample, to protect it pollinating insects.

In certain aspects, the height of a cotton plant is measured from thecotyledon to the topmost structure of the plant. As used herein for acotton plant, measuring to the topmost structure of the cotton plant(also referred to herein as “top of the plant”) means measuring to theuppermost node that is associated with an unfurled leaf at least 1 inchin diameter. One of ordinary skill in the art would recognize that therecan be younger leaves higher up, but that they are curled, and thus itis standard to ignore them when measuring plant height in order ensureconsistent measurements.

On of ordinary skill in the art will recognize that measuring from thecotyledon nodes is more consistent, but the height of a plant can alsobe measured from the top of the soil where the plant is growing to thetopmost structure of the plant as defined above. Without manipulation ofthe soil level intended to obtain a pre-desired result, which isexcluded from a height measurement of this disclosure, one of ordinaryskill in the art would understand that the distance between the top ofthe soil and the cotyledon nodes is about 2 to 6 inches (e.g.,approximately 4 inches on average). Thus, one of ordinary skill in theart would understand that if a measurement of plant height is taken fromthe top of the soil to the top of the plant, that height can beconverted for comparison purposes to the heights disclosed herein bysubtracting 4 inches, i.e., that average distance between the top of thesoil and the cotyledon nodes. For example, if a plant is measured fromthe top of the soil to the top of the plant and the height is recordedas 26 inches, for purposes of this disclosure, that plant's height isconsidered 22 inches.

In certain aspects, a plant is a plant having produced seed but beingless than about 30 inches tall, less than about 24 inches tall, lessthan about 18 inches tall, less than about 15 inches tall, or less thanabout 12 inches tall. In certain aspects, a plant is a plant havingproduced seed but being from any of about 6 inches tall, 12 inches tall,15 inches tall, 18 inches tall, or 24 inches tall to any of about 12inches tall, 15 inches tall, 18 inches tall, 24 inches tall, or 30inches tall (one of ordinary skill in the art would understand how toconvert inches into other units such as centimeters or meters). While itis understood that a plant growing from a seed will start out, forexample, less than 12 inches tall, the growing plant during itsvegetative stage will not have produced seeds. By the time that a plantgrown under normal growth conditions reaches it reproductive stage andproduces seed, it will be taller than, for example, 12 inches tall.

In certain aspects, the plant is a cotton plant having produced at leastone cotton boll that contains a seed, wherein the seed comprises atleast one embryo with a cotyledon in the primordia stage, but being lessthan about 30 inches tall, less than about 24 inches tall, less thanabout 18 inches tall, less than about 15 inches tall, or less than about12 inches tall. In certain aspects, the plant is a cotton plant havingproduced at least one cotton boll that contains a seed, wherein the seedcomprises at least one embryo with a cotyledon in the primordia stage,but being from any of about 6 inches tall, 12 inches tall, 15 inchestall, 18 inches tall, or 24 inches tall to any of about 12 inches tall,15 inches tall, 18 inches tall, 24 inches tall, or 30 inches tall. Whileit is understood that a cotton plant growing from a seed will start out,for example, less than 12 inches tall, the growing cotton plant duringits vegetative stage will not have produced seeds. By the time that acotton plant grown under normal growth conditions produces at least onecotton boll that contains a seed, it will be taller than, for example,12 inches tall.

In certain aspects, a plant is less than about 30 inches tall, less thanabout 24 inches tall, less than about 18 inches tall, less than about 15inches tall, or less than about 12 inches tall but has at least 20seeds, at least 30 seeds, at least 40 seeds, at least 50 seeds, or atleast 60 seeds.

In certain aspects, the plant is a cotton plant that is less than about30 inches tall, less than about 24 inches tall, less than about 18inches tall, less than about 15 inches tall, or less than about 12inches tall but has at least 20 seeds, at least 30 seeds, at least 40seeds, at least 50 seeds, or at least 60 seeds.

Certain aspects provide for growing a population of plants comprisingplants with an altered morphology, wherein the plants in the populationare grown according to the any of the methods disclosed herein. Incertain aspects, at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or99% of the plants in the population produce seed. In certain aspects, atleast one plant of the population is of a reduced height compared to acontrol plant. In certain aspects, at least about 50%, 60%, 70%, 80%,90%, 95%, 98%, or 99% of the plants in the population are of a reducedheight compared to a control plant. In certain aspects, the populationis grown in a controlled environment. In certain aspects, at least about50%, 60%, 70%, 80%, 90%, or 95% of the plants in the population are lessthan about 30 inches tall, less than about 24 inches tall, less thanabout 18 inches tall, less than about 15 inches tall, or less than about12 inches tall. In certain aspects, at least about 50%, 60%, 70%, 80%,90%, or 95% of the plants in the population are from any of about 6inches tall, 12 inches tall, 15 inches tall, 18 inches tall, or 24inches tall to any of about 12 inches tall, 15 inches tall, 18 inchestall, 24 inches tall, or 30 inches tall. In certain aspects, the averageheight of a plant in the population is less than about 30 inches tall,less than about 24 inches tall, less than about 18 inches tall, lessthan about 15 inches tall, or less than about 12 inches tall. In certainaspects, the average height of a plant in the population is from any ofabout 6 inches tall, 12 inches tall, 15 inches tall, 18 inches tall, or24 inches tall to any of about 12 inches tall, 15 inches tall, 18 inchestall, 24 inches tall, or 30 inches tall. In certain aspects, at leastabout 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the plants in thepopulation have at least 20 seeds, at least 30 seeds, at least 40 seeds,at least 50 seeds, or at least 60 seeds. In certain aspects, the plantis a cotton plant and at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%,or 99% of the plants in the population have reached a stage in theirlife cycle wherein pinhead squares have formed.

In certain aspects, the population of plants is grown at an averagedensity of at least 10 plants per square meter. In certain aspects, thepopulation of plants is grown at an average density of at least 30plants per square meter. In certain aspects, the population of plants isgrown at an average density of at least about 60 plants per squaremeter. In certain aspects, the population of plants is grown at anaverage density of at least 67 plants per square meter. In certainaspects, the population of plants is grown at an average density of atleast 75 plants per square meter. In certain aspects, the population ofplants is grown at an average density of at least 100 plants per squaremeter. In certain aspects, the population of plants is grown at anaverage density of at least 125 plants per square meter. In certainaspects, the population of plants is grown at an average density of atleast 150 plants per square meter. In certain aspects, the population ofplants is grown at an average density of about 10, 30, 60, 75, 100, 125,or 150 plants per square meter or any range in between.

In certain aspects, the population of plants comprises at least about 50cotton plants, at least about 100 cotton plants, at least about 500cotton plants, at least about 1,000 cotton plants, at least about 5,000cotton plants, at least about 10,000 cotton plants, at least about100,000 cotton plants, at least about 500,000 cotton plants, at leastabout 1,000,000 cotton plants. Similarly, in certain aspects, thepopulation of plants comprises 50 or more cotton plants, 100 or morecotton plants, 500 or more cotton plants, 1,000 or more cotton plants,5,000 or more cotton plants, 10,000 or more cotton plants, 100,000 ormore cotton plants, 500,000 or more cotton plants, or 1,000,000 or morecotton plants.

Certain aspects provide for a cotton plant grown under any of thestressed conditions disclosed herein, and grown as a member of apopulation of cotton plants, wherein the cotton plant is grown in thepopulation with an average density of at least 10, 30, 60, 67, 75, 100,125, or 150 plants per square meter, for at for at least 40 days, for atleast 50 days, for at least 60 days, for at least 70 days, or for atleast 80 days during the time from when the cotton seed germinates towhen the cotton plant produces at least one cotton boll that contains aseed, wherein the seed comprises at least one embryo with a cotyledon inthe primordia stage.

Plant Breeding

In addition to growing a plant, such as to produce a plant with analtered morphology and/or produce a seed, methods provided herein alsoapply the methods of growing a plant for breeding purposes. In certainaspects, at least one seed is collected from a plant grown by at leastone of the aforementioned methods and/or having one of theaforementioned morphologies. In certain aspects, the collected seed orplant grown from the seed can be tested (also referred to as “screened”)for traits. In certain aspects, the seed is tested by genotyping and/orphenotyping. In certain aspects, the information obtained by testing isused as the basis for growing and/or crossing a plant or otherwise usedin a plant breeding program.

Commercial plants, such as cotton, are generally grown in open fieldsbecause the space needed to grow large populations of plants. Oneadvantage of growing plants with an altered morphology, in particularplants with reduced height compared to control plants, is that shorterplants tend to also be more compact overall, i.e., they take up lessspace. This attribute—as well as the potential of using a reduced amountof soil volume per plant—allows plants to be grown at higher densitiesthan recommended or considered viable to produce plants capable of seedproduction. Under normal planting densities, a population of 1000 cottonplants requires about 40,000 square meters of space. Using the methodsdescribed herein that reduce the size of the plants, the same sizepopulation can be grown with only about 4,800 square meters of space.This makes it much more feasible to grow large populations of plants incontrolled environments, such as to exclude pollinating insect that mayinterfere with crosses made for plant breeding. In certain aspects, aplant is grown as part of a population of plants where the averagedensity of plants is at least 10 plants per square meter. In certainaspects, a plant is grown as part of a population of plants where theaverage density of plants is at least 30 plants per square meter. Incertain aspects, a plant is grown as part of a population of plantswhere the average density of plants is at least 60 plants per squaremeter. In certain aspects, a plant is grown as part of a population ofplants where the average density of plants is at least 67 plants persquare meter. In certain aspects, a plant is grown as part of apopulation of plants where the average density of plants is at least 75plants per square meter. In certain aspects, a plant is grown as part ofa population of plants where the average density of plants is at least100 plants per square meter. In certain aspects, a plant is grown aspart of a population of plants where the average density of plants is atleast 125 plants per square meter. In certain aspects, a plant is grownas part of a population of plants where the average density of plants isat least 150 plants per square meter. In certain aspects, a plant isgrown as part of a population of plants where the average density isabout 10, 30, 60, 75, 100, 125, or 150 plants per square meter or anyrange in between. Certain aspects provide for such a population ofplants wherein the population comprises plants with an alteredmorphology and/or grown according to the methods provided herein.

A population of plants with an average density disclosed anywhere hereinmay be relatively small to very large. In certain aspects, thepopulation of plants comprises at least about 50 cotton plants, at leastabout 100 cotton plants, at least about 500 cotton plants, at leastabout 1,000 cotton plants, at least about 5,000 cotton plants, at leastabout 10,000 cotton plants, at least about 100,000 cotton plants, atleast about 500,000 cotton plants, at least about 1,000,000 cottonplants. Similarly, in certain aspects, the population of plantscomprises 50 or more cotton plants, 100 or more cotton plants, 500 ormore cotton plants, 1,000 or more cotton plants, 5,000 or more cottonplants, 10,000 or more cotton plants, 100,000 or more cotton plants,500,000 or more cotton plants, or 1,000,000 or more cotton plants. Thepopulation may be grown under controlled conditions. In certain aspects,at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the plantsin the population are at their reproductive stage. In certain aspects,at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the plantsin the population produce seed. In certain aspects, the plant is acotton plant and at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or99% of the cotton plants in the population have reached a stage in theirlife cycle wherein pinhead squares have formed.

In certain aspects, at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99%of the plants in the population produce at least 20 seeds, at least 30seeds, at least 40 seeds, at least 50 seeds, or at least 60 seeds. Incertain aspects, at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% ofthe plants in the population are less than about 30 inches tall, lessthan about 24 inches tall, less than about 18 inches tall, less thanabout 15 inches tall, or less than about 12 inches tall. In certainaspects, the average height of the population is less than about 30inches tall, less than about 24 inches tall, less than about 18 inchestall, less than about 15 inches tall, or less than about 12 inches tall.

Under normal commercial planting conditions, a cotton plant producesfrom about 18 to about 30 nodes, with the first fruiting node betweennode 5 and 9. When plants are grown under the stressed conditionsdisclosed herein, bolls typically set at nodes 5 or 6, one boll perplant.

Types of Plants

Unless otherwise specified, this disclosure is not limited to anyparticular type of plant. In certain aspects, the plant is a crop plant.Representative examples of crop plants include: a corn or maize plant, asoybean plant, a canola plant, a cotton plant, a wheat plant, and asugarcane plant. In certain aspects, the plant is a cotton plant. Inanother aspect, a plant provided herein is selected from the groupconsisting of Acacia plant, an alfalfa plant, an aneth plant, an appleplant, an apricot plant, an artichoke plant, an arugula plant, anasparagus plant, an avocado plant, a banana plant, a barley plant, abean plant, a beet plant, a blackberry plant, a blueberry plant, abroccoli plant, a Brussels sprout plant, a cabbage plant, a canolaplant, a cantaloupe plant, a carrot plant, a cassava plant, acauliflower plant, a celery plant, a Chinese cabbage plant, a cherryplant, a cilantro plant, a citrus plant, a clementine plant, a coffeeplant, a corn plant, a cotton plant, a cucumber plant, a Douglas firplant, an eggplant plant, an endive plant, an escarole plant, aneucalyptus plant, a fennel plant, a fig plant, a forest tree plant, agourd plant, a grape plant, a grapefruit plant, a honey dew plant, ajicama plant, kiwifruit plant, a lettuce plant, a leek plant, a lemonplant, a lime plant, a Loblolly pine plant, a mango plant, a maple treeplant, a melon plant, a mushroom plant, a nectarine plant, a nut plant,an oat plant, an okra plant, an onion plant, an orange plant, anornamental plant plant, a papaya plant, a parsley plant, a pea plant, apeach plant, a peanut plant, a pear plant, a pepper plant, a persimmonplant, a pine plant, a pineapple plant, a plantain plant, a plum plant,a pomegranate plant, a poplar plant, a potato plant, a pumpkin plant, aquince plant, a radiata pine plant, a radicchio plant, a radish plant, arapeseed plant, a raspberry plant, a rice plant, a rye plant, a sorghumplant, a Southern pine plant, a soybean plant, a spinach plant, a squashplant, a strawberry plant, a sugar beet plant, a sugarcane plant, asunflower plant, a sweet corn plant, a sweet potato plant, a sweetgumplant, a tangerine plant, a tea plant, a tobacco plant, a tomato plant,a turf plant, a vine plant, watermelon plant, a wheat plant, a yamplant, and a zucchini plant.

The plant may be any whole plant, or part of a plant, or tissue culturederived from a plant, or plant seed. In certain aspects, the plant is acotton plant, a maize plant, or a soybean plant. A cotton plant for usein methods described herein can be at any of various developmentalstages.

Table 1 describes different cotton developments stages.

TABLE 1 Accumulated DD60s Heat Units Previous Developmental Stage DaysMethods New Methods* Planting to Emergence 4 to 9 50 to 60  76-171Emergence to First Square 27 to 38 425 to 475 513-722 Square to Flower20 to 25 300 to 350 380-475 Planting to First Flower 60 to 70 775 to 8501140-1330 *based on a hypothetical example with consistent daily hightemperatures of 90° F. (32.2° C.) and consistent daily low temperaturesof 68° F. (20° C.).

DD60s are the accumulated heat units a plant receives each day and aredetermined by averaging each day's maximum and minimum temperatures andthen subtracting 60, i.e. DD60 =((° Fmax+° Fmin)/2) -60. The DD60scalculated for each day are added to determine the total DD60s for agiven number of growing days.

Flower Timing

Within a population of plants, even for plants planted at the same time,the time at which they mature and/or start to flower and/or produceseeds is not necessarily simultaneous. For example, FIGS. 5 and 6 show adistribution of when standard cotton plants in a population start toflower, starting with just a small percentage in the first few days,then increasing over several days, and then the numbers tail off (FIG.5) until 100% have flowered (cumulative shown in FIG. 6). In currentmethods of growing cotton plants under standard conditions, the time offlowering and seed maturation can be spread across a number of days oreven weeks. Thus, this may require multiple rounds of gathering samplesif it is desired to gather the samples at the same point of maturationthroughout the population.

It has been discovered that for a population of plants grown accordingto methods utilizing the stressed conditions disclosed herein and/orhaving the resultant reduced statute morphology, the window of floweringtime of the plants in the population is more synchronized (narrower)compared to control plants grown under standard conditions. That is, thewindow of time from when the first plants in the population begin toflower and/or produce seed and when a significant percentage,substantially all, or all of the plants in the population have floweredand/or produced seed is shorter. For example, in certain aspects,cumulatively 50%, 60%, 70%, 75%, 80%, 90%, 95%, 98%, 99%, or 100% ofplants in a population of cotton plants disclosed herein have floweredand/or produced seed within 1 day , 2 days, 3 days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14days from when the first plant in population flowers and/or producesseed. For example, in certain aspects, cumulatively 50%, 60%, 70%, 75%,80%, 90%, 95%, 98%, 99%, or 100% of plants in a population of cottonplants disclosed herein have flowered and/or produced seed within 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75days after planting. In certain aspects, the timespan between when thefirst plant in a population of cotton plants begins to flower and/orproduce seed and cumulatively 50%, 60%, 70%, 75%, 80%, 90%, 95%, 98%,99%, or 100% of plants in a population have flowered and/or producedseed is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20%, 25%,30%, 33%, 35%, 40%, 50%, 60%, 70%, 75%, or 80% less time in comparisonto a control population of plants. As disclosed herein, the populationof plants comprises at least about 50 cotton plants, at least about 100cotton plants, at least about 500 cotton plants, at least about 1,000cotton plants, at least about 5,000 cotton plants, at least about 10,000cotton plants, at least about 100,000 cotton plants, at least about500,000 cotton plants, at least about 1,000,000 cotton plants.Similarly, in certain aspects, the population of plants comprises 50 ormore cotton plants, 100 or more cotton plants, 500 or more cottonplants, 1,000 or more cotton plants, 5,000 or more cotton plants, 10,000or more cotton plants, 100,000 or more cotton plants, 500,000 or morecotton plants, or 1,000,000 or more cotton plants.

It is contemplated that tighter synchronization of maturation,flowering, and/or seed production timing will permit breeders to moreconsistently predict when plants in a population will flower and/orproduce seeds. This reduced variability in developmental timing willimprove operations and timing in, for example, industrial breedingprograms, thereby reducing overall cycle time and resource waste.

EXAMPLES

The following examples are included to demonstrate certain aspects ofthe invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific aspects which are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the invention.

Example 1 Cotton Plants Grown Under High Stress and High Density

Approximately 188,000 plant pots, each comprising dimensions ofapproximately 7 cm wide×approximately 7 cm long×approximately 8 cm deep(for a total interior volume of slightly less than 400 mL) wereassembled and prepared to receive growth media and seeds. Into each pot,approximately 380 mL of wet peat moss media was placed, along withOsmocote fertilizer following the manufacturer's recommendations. Intoeach pot, 2-3 F2 seeds of a certain cotton hybrid variety were planted.

Pots containing soil and seeds were then arranged into high-densitygrowing blocs, each bloc comprising 324 pots in a 4.8 m² area. 580 ofthese blocs were squeezed into a shade house, such that a total of187,920 pots comprised a total of 2,784 m² controlled environment space.The pots were arranged into this growing density immediately after seedswere planted and from that point on, the plants were subjected to thefollowing temperature and irrigation stress conditions, in addition tothe reduced soil volume and/or population density.

High Temperature. Due to the prevailing climate of the latitude andregion where the greenhouse was located (Costa Rica), the temperaturevariation in any 24-hour photocycle remained predictable throughout thegrowing period. During each 24-hour day, the temperatures would risefrom nighttime lows of 18-22° C., rapidly reaching 30° C. in the morningwhen the greenhouse was in direct sun, and continuing to increase to anafternoon maximum of 35-45° C., which was maintained for at least 2-12hours, each day. Temperatures dropped in the evenings, especially afterthe sunset, to eventually return to a nighttime low, which typicallyoccurred between 1 a.m. and 4 a.m.

Reduced Irrigation. Plants were irrigated via drip lines so that thevolume of water accessible to the plant over any period of time could becarefully metered and controlled. Irrigation was provided throughoverhead sprinklers. To determine and control the amount of irrigationeach plant received, several samples of plants were weighed before andafter each irrigation. The weight gained was converted to volume ofwater and then divided by the total number of plants in the sample todetermine the average amount of water each plant had received. Throughcareful weighing and sampling, this system was calibrated so that anaverage volume of water could be reliably, and verifiably, delivered toeach plant in the covered environment. Through extensive testing acrossmany growing cycles, it was discovered that approximately 100 mL ofwater per plant per 24-hour period was sufficient to keep the plantalive and induce the desired miniaturized morphology.

After approximately 3 days after planting (DAP), seedlings began toemerge from the pots. As emergence and growth proceeded, some potsproduced more than one seedling (2-3 seeds were planted/pot to ensure atleast one seedling/pot would be recovered), so additional seedlings wereremoved from those pots so that all pots produced only one seedling perpot (“doubles” and “triples” were culled down to singles). Thus, a totalof 187,920 plants were now growing within a total of 2,784 m² within thecontrolled environment. During the next few days, plants were allowed toself-pollinate.

At 20 DAP, a plant treatment agent comprising mepiquat was applied.Approximately 0.5 L of PIX® per hectare was applied to the plants.Application comprised homogenizing 139 mL of PIX® in 20 L of water andloading the mixture into a backpack sprayer and then using the sprayerto apply the diluted PGR to the approximately 188,000 plants growing inthe controlled environment. This same treatment was reapplied to theplants three more times in the following weeks, up until 60 DAP.

It was noted that the total volume of liquid added to the plants in anyone day that the PGR was applied amounted to approximately 0.5 mL perplant, and so was considered a negligible amount of additionalirrigation beyond the 100 mL that was meted out to each plant each day.

At approximately 25 DAP, at least one pinhead square appeared on all ofthe plants, typically at the 5^(th) node. By 46-50 DAP, most plants haddeveloped fully-formed flowers and by 60 DAP, all plants had flowers.Between 86-100 DAP, fully-mature bolls were harvested from the plantsand the seeds within recovered. Seed yield was typically 20-30 seeds perboll.

In this example, at least 20 seeds were recovered from each of the187,920 plants, all of which were cultivated within a 2,784 m² area ofcontrolled environment. Producing seeds from this same number of crossesusing current research methods, wherein cotton plants are typicallyplanted at approximately 10 plant/m² in the field, would requireapproximately 18,800 m², or 1.8 hectares. Thus, the methods describedherein allow one to produce seeds from the same number of crosses usingonly 10.5% of the growing area of current protocols.

Table 3a-g below shows data comparing field grown plants not subjectedto stressed conditions in comparison to mini-cotton plants grownaccording to stressed conditions disclosed herein. At both early andlate times, plants grown under the stressed conditions weresignificantly smaller in all aspects measured than field grown plants.

TABLE 3a Days After Average of Height Planting Number from Cotyledon toRow Labels (DAP) of Plants Last open leaf (cm) Early Field plant-PIXapplication 34 10 Field plant-PIX application 21.95 Mini plants-PIXapplication 17 5 Mini plants-PIX application 9.8 Late Field Plant-NearCut off 86 5 Field Plant-Near Cut off 162.4 Mini Plant Harvest 119 5Mini Plant Harvest 29.3

TABLE 3b Average of # Average of # Nodes Bolls Early Field plant-PIXapplication 8.7 Field plant-PIX application n/a Mini plants-PIXapplication 3 Mini plants-PIX application n/a Late Field Plant-Near Cutoff 26.8 Field Plant-Near Cut off 21.2 Mini Plant Harvest 8.6 Mini PlantHarvest 1.2

TABLE 3c Average of 1st Average of Weight Node with boll from N0 and up(grams) Early Field plant-PIX application 5.4 Field plant-PIXapplication 25.67 Mini plants-PIX application 0 Mini plants-PIXapplication 6.2 Late Field Plant-Near Cut off 5.6 Field Plant-Near Cutoff 1028 Mini Plant Harvest 5.6 Mini Plant Harvest 18.1

TABLE 3d Average of Flowers/ Average of Flowers/ Fruits-Fresh WeightFruits-Dry Weight at site (grams) at lab (grams) Early Field plant-PIXapplication 0.21 Field plant-PIX application 0.045 Mini plants-PIXapplication 0 Mini plants-PIX application 0 Late Field Plant-Near Cutoff 307.44 Field Plant-Near Cut off 46.6 Mini Plant Harvest 6.58 MiniPlant Harvest 5.55

TABLE 3e Average of Leaves- Average of Leaves- Fresh Weight at site DryWeight at lab (grams) (grams) Early Field plant-PIX application 18.72Field plant-PIX application 2.679 Mini plants-PIX application 3.68 Miniplants-PIX application 0.14 Late Field Plant-Near Cut off 281.84 FieldPlant-Near Cut off 65.9 Mini Plant Harvest 5.92 Mini Plant Harvest 1.296

TABLE 3f Average of Stem-Fresh Average of Stem-Dry Weight at site(grams) Weight at lab (grams) Early Field plant-PIX application 4.91Field plant-PIX application 0.363 Mini plants-PIX application 0.74 Miniplants-PIX application 0.062 Late Field Plant-Near Cut off 342.16 FieldPlant-Near Cut off 90.66 Mini Plant Harvest 5.16 Mini Plant Harvest1.704

TABLE 3g Ave. dry weight total (grams) (sum of above dry weights) EarlyField plant-PIX application 3.087 Mini plants-PIX application 0.202 LateField Plant-Near Cut off 203.16 Mini Plant Harvest 8.55

1-56. (canceled)
 57. A population of cotton plants with an alteredmorphology, wherein the population is in a controlled environment,wherein the average density of the cotton plants in the population is atleast 10 plants per square meter and the plants at the density are in anaverage volume of growth media of less than about 1,000 mL per plant,and wherein at least about 70% of the individual cotton plants in thepopulation are between about 12 inches and 24 inches tall and have atleast one boll that contains at least 20 seeds.
 58. The population ofclaim 57, where the average density of the plants in the population isat least 30 plants per square meter.
 59. The population of claim 57,wherein the plants are at the density in an average volume of growthmedia of less than about 500 mL per plant.
 60. The population of claim57, wherein at least about 80% of the individual cotton plants in thepopulation are between about 12 inches and 24 inches tall and have atleast one boll that contains at least 20 seeds.
 61. The population ofclaim 57, wherein at least about 70% of the individual cotton plants inthe population are between about 12 inches and 18 inches tall and haveat least one boll that contains at least 20 seeds.
 62. The population ofclaim 61, wherein at least about 80% of the individual cotton plants inthe population are between about 12 inches and 18 inches tall and haveat least one boll that contains at least 20 seeds.
 63. The population ofclaim 57, wherein the population comprises at least about 1,000 cottonplants.
 64. The population of claim 63, wherein the population comprisesat least about 10,000 cotton plants.
 65. The population of claim 64,wherein the population comprises at least about 100,000 cotton plants.66. The population of claim 57, wherein the controlled environment issubstantially free of pollinating insects.
 67. The population of claim57, wherein the population of plants is grown at a temperature of atleast about 36° C. for at least about 2 hours per day, for at least 50days during the time from the average day of germination of the cottonseeds of the population to the average day that the plants produce atleast one cotton boll that contains a seed.
 68. The population of claim67, wherein the population of plants are grown at a temperature of atleast about 37° C. for at least about 2 hours per day, for at least 50days during the time from the average day of germination of the cottonseeds of the population to the average day that the plants produce atleast one cotton boll that contains a seed.
 69. The population of claim57, wherein the population of plants is grown while providing less thanabout 400 mL of water per day on average, for at least 50 days duringthe time from the average day of germination of the cotton seeds of thepopulation to the average day that the plants produce at least onecotton boll that contains a seed.
 70. The population of claim 69,wherein the population of plants are grown while providing less thanabout 300 mL of water per day on average, for at least 50 days duringthe time from the average day of germination of the cotton seeds of thepopulation to the average day that the plants produce at least onecotton boll that contains a seed.
 71. The population of claim 57,wherein the population of plants is grown at a temperature of at leastabout 36° C. for at least about 2 hours per day, for at least 50 daysduring the time from the average day of germination of the cotton seedsof the population to the average day that the plants produce at leastone cotton boll that contains a seed and growing the cotton plants whileproviding less than about 400 mL of water per day on average, for atleast 50 days during the time from the average day of germination of thecotton seeds of the population to the average day that the plantsproduce at least one cotton boll that contains a seed.
 72. Thepopulation of claim 57, wherein at least about 80% of the individualcotton plants in the population are between about 12 inches and 24inches tall and have at least one boll that contains at least 20 seeds.73. The population of claim 57, wherein a plant growth regulatorselected from the group consisting of chlormequat-CL, mepiquat-CL,AMO-1618, clorphonium-Cl, tetcylacis, ancymidol, flurprimidol,paclobutrazol, uniconazole-P, inabenfide, prohexadione-CA,trinexapac-ethyl, daminozide, exo-16,17-, dihydro-GA5-13-acetate,systemin, phytosulfokine, and rapid alkalinization factor is applied tothe population of plants.
 74. The population of claim 73, wherein theplant growth regulator is mepiquat-CL.
 75. The population of claim 73,wherein about 0.5 mL of the plant growth regulator is applied per plant.76. The population of claim 73, wherein the plant growth regulator isapplied about 20 days after planting.